JP3536109B2 - Foods composed of microorganisms containing high concentration omega-3 polyunsaturated fatty acids - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a lipid containing a high concentration of omega-3 highly unsaturated fatty acid (HUFA) suitable for use as a heterotrophic organism and as a food additive for humans and animals, or in pharmaceuticals and industrial products. The present invention relates to a heterotrophic living organism for producing a bacterium and a method for culturing the same.

[0002]

2. Description of the Related Art Omega-3 highly unsaturated fatty acids (HUFA)
s) prevent arteriosclerosis and heart disease, reduce inflammation,
It has recently been recognized as an important nutritional compound in suppressing the growth of tumor cells and is of great commercial interest. These advantages derive from omega-3 HUFAs that cause competitive inhibition of compounds produced from omega-6 fatty acids, as well as omega-3 HUFs.
It also derives from advantageous compounds produced directly from As itself (Simopoulos et al.). Omega 6 fatty acids are the predominant HUFAs found in plants and animals.
Currently, the commercial nutritional source of Omega-3 HUFAs is certain fish oils that can contain 20-30% of these fatty acids. The effects of these fatty acids are obtained by eating fish several times a week or ingesting concentrated fish oil daily. Therefore, a large amount of fish oil is processed, encapsulated and sold every year as a nutritional supplement. However, these fish oil supplements have some important problems, including the bioaccumulation of fat-soluble vitamins and high concentrations of saturated omega-6 fatty acids, both of which It can harm your health.

Another source of Omega-3 HUFAs is the microbiota Thraustochytrium and Schizochytrium, which are detailed in related US Pat. No. 5,130,242. These microbiota are heterotrophic and have high concentrations of Omega-3 HUFAs.
Has the advantage that However, there remains a need to improve fermentation methods for these microflora and to identify ways to use improved forms of microflora products.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a microorganism that produces a high concentration of omega-3 highly unsaturated fatty acids and a method for growing the same. To do.

[0005]

In order to achieve the above-mentioned object, the invention according to claim 1 comprises: a) a long chain omega-3 highly unsaturated fatty acid, and a genus Thraustochytriales, A microorganism selected from the genus Schizochytrium and mixtures thereof, and b) containing short chain omega-3 highly unsaturated fatty acids and selected from the group consisting of flaxseed, rapeseed, soybeans, avocadomile and mixtures thereof Foods consisting of substances are the gist.

According to a second aspect of the present invention, in the food according to the first aspect, the food contains about 5 to 95% by weight of
The summary is that the substances listed above are included.

According to a third aspect of the invention, in the food according to the first aspect , the food is an extruded product.
Is the gist.

[0008] inventions as set forth in claim 4, a) a long chain tail bud of
Moth containing highly unsaturated fatty acids, and
Triales, Schizochytrium and their mixtures?
Fatty acids obtained from microorganisms selected from
Contains omega-3 highly unsaturated fatty acids, and flaxseed,
Made from rapeseed, soybeans, avocado meal and mixtures thereof
Consisting of fatty acids obtained from materials selected from the group
The main point is food.

[0009]

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016]

One embodiment of the present invention relates to a novel method of growing the microbial flora Traustochytrium, Schizochytrium and mixtures thereof. The method involves growing the microflora, especially in a medium having non-chloride sodium salts such as sodium sulfate. More specifically, most of the sodium needed for fermentation is supplied as non-chloride sodium salts. The method of the present invention is particularly useful for commercial production because the chloride content in the medium is significantly reduced, thus avoiding chloride corrosion of the fermentor. In addition, the present invention is particularly useful in the manufacture of food products utilized in aquaculture. It was found that Traustochytrium and Schizochytrium cultivated in this type of medium formed much smaller clamps than when cultivated in high chloride medium, thus making it more accessible as a dietary source for shrimp larvae. This is because In particular, Thraustochytrium and Schizochytrium grown in sodium sulphate containing medium may have cell aggregates with an average size of about 150 microns or less in diameter.

In a further embodiment of the present invention, a microflora biomass is produced which comprises a cell aggregate having an average size of less than about 150 microns, such as Thraustochytrium, Schizochytrium and mixtures thereof. Microbiota biomass is useful for aquaculture, especially because it has a high sterol content and a high omega tri-unsaturated fatty acid (HUFA) content. Useful for feeding shrimp). In addition, the microflora, due to the small size of cell aggregates, can be ingested by offspring, brine shrimp, stink bugs and mollusks. The invention also includes a method of producing these organisms, which method comprises feeding a genus Thraustochytrium, a schizochytrium and a mixture thereof having an average cell size of about 150 microns or less.

A further embodiment of the present invention is a microflora selected from the group consisting of Traustochytrium, Schizochytrium and mixtures thereof, and flaxseed, rapeseed, soybean,
A food product comprising an avocado meal and an additive ingredient selected from the group consisting of mixtures thereof. This food is particularly effective in that it contains a large amount of long-chain omega-3 fatty acids and short-chain omega-3 fatty acids derived from the additive components. In a further embodiment, the food product is produced by extrusion. The extruding step may include the step of mixing the microflora with the additive components, which may reduce the water content of the food product. The food is then heated and extruded, thus removing most of the reduced water content. The residual amount of the original water content is easily removed by natural drying or short-time calcination to reduce the total energy required for drying, and by extending the high temperature drying, the degree of degradation of Omega-3 HUFAs is reduced. It

[0020]

DETAILED DESCRIPTION OF THE INVENTION As a definition throughout this specification, a fatty acid is understood to be an aliphatic monocarboxylic acid. A lipid is understood to be a fat or oil containing glyceride esters of fatty acids with related phosphatides, sterols, alcohols, hydrocarbons, ketones and related compounds.

In this specification, the structure of fatty acid is
Uses a commonly used shorthand system (example
For example, Weete, 1980). With this system
Starts with the letter “C” and indicates the number of carbon atoms in the hydrocarbon chain
Followed by a colon and a number indicating the number of double bonds,
That is, it becomes C20: 5 eicosapentaenoic acid. fatty acid
Are numbered starting with carboxy carbon
There is. Double bond position is added with Greek letter Delta (Δ)
, Followed by the number of carbon atoms in the double bond.
That is, C20: 5 Omega 3 system Δ^5,8,11,14,17Becomes
The "Omega" notation is a shorthand notation system for unsaturated fatty acids.
And the numbering from the carboxyl terminal carbon is used.
I have been. For convenience, the "Omega 3 system" was encoded.
Therefore, in particular, use the numerical expression shorthand notation described in this specification.
N-3 is used when Omega 3 system highly unsaturated
The fatty acid is polyethylene fatty acid, and the
When it is derived from the terminal methyl group of fatty acid,
It is to be understood as containing three carbon atoms. Yo
, Eicosapenta, a highly unsaturated omega-3 fatty acid
When enoic acid is completely expressed, C20: 5n-3Δ
^5,8,11,14,17Becomes For simplification, double bond position (Δ
^5,8,11,14,17) Is omitted. Eicosapentaenoic acid
C20: 5n-3, and docosapentaenoic acid (C2
2: 5n-3Δ^{7,10,13,16 , 19}) Is C22: 5n-3
Yes, docosahexaenoic acid (C22: 6n-3Δ^{4,7
, 10,13,16,19}) Is C22: 6n-3. "Highly tired
The term "Japanese fatty acid" is a fat containing four or more double bonds.
It is a fatty acid. "Saturated fatty acid" is 1-3 double bonds
It is a fatty acid having a combination.

A collection and selection method has been created for the easy isolation of many microbial strains with the following combinations of economically desirable properties for producing Omega-3 HUFAs. It is capable of 1) heterotrophic growth, 2) high omega-3 HUFAs content, 3) unicellular, 4) preferably saturated, and low omega-6 HUFAs content, 5) preferably colorless. , White or essentially colorless cells, 6) preferably thermostable (ability to grow at temperatures above 30 ° C.) and 7) preferably broad salinity (growth over a wide range of salinity, especially low salinity) It is possible). This method is detailed in related US Pat. No. 5,130,242.

Using this collection and selection method, the total dry weight percent (% dwt) of the cells has a fatty acid content of up to about 45%, is grown over a temperature range of 15 to 48 ° C., and has a salinity. Single cell microbiota strains that grow in very low media can be isolated. Many of the very high strains of Omega-3 are very slow growing. Strains isolated by the method outlined above and exhibiting rapid growth rate, excellent productivity and high Omega-3 HUFA content have an Omega-3 unsaturated fatty acid content of up to about 12% dwt. ing.

In one embodiment of the present invention, a Thraustochytrium sp., Schizochytrium sp. And a mixture thereof having a high omega-3 HUFA content are grown in a fermentation medium containing a non-chloride sodium salt, preferably sodium sulfate. More specifically, most of the sodium needed for fermentation is supplied as non-chloride sodium salts. For example,
About 75% or less, preferably about 50% or less, more preferably about 25% or less of sodium in the fermentation medium is supplied as sodium chloride. A particular advantage of the present invention is that, in the absence of large amounts of chloride, which can corrode the vessel in which the microflora is grown as well as other fermentation or downstream processing equipment,
The medium provides the source of sodium necessary for the growth of the microflora. Surprisingly, the microflora of the present invention is less than about 3 g / l, preferably less than about 500 mg / l, more preferably about 250 mg / l.
The following is more preferably viable at a chloride concentration of about 60 to 120 mg / l and about 50% per sugar.
It was found that the above biomass is produced in large quantities.
Further advantages of the present invention are as follows.
Despite its high FA content, it is to produce a microflora with cell aggregates of a size small enough for ingestion by juvenile shrimp, saltwater shrimp, pit viper and mollusks.

Among the non-chloride sodium salts are soda ash (mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulphate and mixtures thereof, of which sodium sulphate is preferred. Soda ash, sodium carbonate and sodium bicarbonate have the property of increasing the pH of the fermentation medium and thus require control steps to maintain the pH of the medium properly.
Sodium sulphate concentration is effective to meet the salinity requirements of the microflora, preferably sodium concentration (g of Na / g
1) is about 1.0 g / l or more, more preferably about 1.0 to 50.0 g / l, even more preferably about 2.0.
~ 25 g / l.

Surprisingly, the non-chloride sodium salt,
In particular, it has been found that fermentation of the strain in the presence of sodium sulfate limits the size of the cell aggregates of the strain to about 150 microns or less, preferably about 100 microns or less, more preferably about 50 microns or less. Was issued. The term cell aggregate size, as used herein, refers to the approximate average size of the clumps, or aggregates, of cells in the fermentation medium of a microflora culture. Usually, about 25% or more, preferably about 50% or more, and more preferably about 75% or more of the cell aggregates in the microflora culture have cell aggregates having an average size or less. The microbiota cells produced according to the present invention are resuspended in a liquid state by a blender, a vortex generator or the like, that is, even when physically stirred, not only after the coagulation and / or drying of the biomass but also the fermentation medium. The dimensional parameters of the above-mentioned cell aggregates are satisfied even when the cell aggregates are present in. The method of the present invention is particularly applicable to a microflora that replicates by continuous mitosis (replication by dividing one cell into two cells, each of the two cells further dividing into two, and so on). It makes sense. It has the property of aggregating forming multicellular aggregates when cells are repeatedly and rapidly exposed to this process,
This is because this aggregate often exceeds the size range of the above cell aggregate. Schizochytrium replicates by continuous mitosis and formation of sporangium that releases sperm. However, the genus Thraustochytrium forms sporangia and replicates only by releasing sperm. Spore
Aggregation can also be a problem in the genus Thraustochytrium, which replicates by forming sperm. This is especially the tendency for individual cells of the genus Thraustochytrium to tend to be larger in size, even though the number of cells in the assembly is not as high as that formed by continuous mitosis, thus consisting of a small number of cells Agglomerates grow larger. However, AT, a deposited strain of the genus Thraustochytrium with less pronounced aggregation
CC 26185 was confirmed.

In another aspect of the present invention, it has been found that the lipid content of the strain can be increased by limiting the oxygen content of the fermentation medium during the growth of Thraustochytrium, Schizochytrium and mixtures thereof. The optimum oxygen concentration for adipogenesis can be determined for any microflora by varying the oxygen content of the medium. In particular, the oxygen content of the fermentation medium is less than about 40% saturation, preferably about 5-40.
Oxygen content of% saturation is maintained.

The growth of the strain according to the method of the present invention can be carried out at any temperature as long as the temperature induces the strain to grow sufficiently. This is, for example, about 5 to 48 ° C, preferably 15 to 40 ° C, more preferably about 25 to 35 ° C.
Is. Usually, if the pH is not adjusted by addition of acid or buffer, the alkalinity of the medium increases during fermentation. Strain p
It grows in the range of H of 5.0 to 11.0, preferably about 6.0 to 8.5.

Various fermentation parameters for inoculation, growth and recovery are detailed in US Pat. No. 5,130,242. The biomass harvested from the fermentation tube is dried (eg, spray dried, tunnel dryin
g), by vacuum drying or similar methods), can be used for feeding, ie feeding, to any animal, whose meat or products are consumed by humans. Similarly, the extracted Omega-3 HUFAs can also be used as a feeding or nutritional supplement. Also, the harvested and washed biomass can be used directly (without drying) as a feed supplement. To extend shelf life, wet biomass is acidified (pH about 3.5-4.5) and / or pasteurized or rapidly heated to inactivate enzymes, and then vacuum or non-oxidizing atmospheres (eg. , N ₂ or CO ₂ ) and can be bottled and packaged. The term "animal" refers to any organism belonging to the animal kingdom and includes, without limitation, any animal from which poultry, seafood, beef, pork or lamb is obtained. Seafood can be obtained from fish, shrimp and shellfish without limitation. The term "product" includes any product other than meat obtained from such animals, including, without limitation, eggs or other products. When omega-3 HUFAs or extracted omega-3 HUFAs in harvested biomass are fed to these animals,
Incorporated in animal meat, eggs or other products to increase the omega-3 HUFA content in these.

In a further embodiment of the invention, the harvested biomass is used as food for shrimp, saline shrimp, pit viper and molluscs, especially shrimp. When I'm still in my childhood,
Shrimp juveniles have a dietary source that is too large to be available. In particular, shrimp larvae are unable to utilize a dietary source with diameters greater than about 150 microns at certain stages of development. Thus, as broadly described above, microflora grown in fermentation media containing non-chloride sodium salts, in particular sodium sulfate, are suitable for use as shrimp foods. As noted above, microbiota cell aggregates grown under such conditions generally have dimensions of about 150 microns or less, preferably about 100 microns or less, more preferably about 50 microns or less. There is.

A further advantage of using the microflora of the present invention as a shrimp feed source is that the microflora contains large amounts of cholesterol-containing sterols, which is a major feeding requirement for shrimp larvae. Generally, the microflora of the present invention are preferably at least about 0.1% ashless dry weight (afdw), more preferably at least about 0.5% a.
fdw, even more preferably at least about 1.0% af
It has a sterol content of dw. In addition, the microbiota of the present invention will typically have a cholesterol content of preferably at least about 15%, more preferably at least about 25%, even more preferably at least about 40% of the total sterol content. In addition, the microflora biomass of the present invention also provides shrimp with additional nutrients such as omega-6 fatty acids, proteins, carbohydrates, pigments and vitamins.

The microbial product of the present invention is an omega-3 HUF of fish, shrimp and other products produced by aquaculture.
It is valuable as a source of As. This product can be used as food, as in the case of the shrimp described above, or added directly as a general feeding supplement to shrimp and fish, or salted shrimp or other live shrimp for ingestion of aquaculture organisms. Can be fed to native feeding organisms. By using this microflora in this way, a shrimp farmer can obtain a shrimp having a significantly high growth rate and / or survival rate, and can produce a more robust shrimp after the childhood. it can.

For most feedings, the fatty acid content of the harvested cells is about 15-50% dwt, and the majority of the remaining material is proteins and carbohydrates. Since some of the evaluated strains have all essential amino acids, the protein plays a major role in the nutritional value of the cell and is considered to be a nutritionally balanced protein.

In a further embodiment of the present invention, a food product comprising the genus Thraustochytrium, Schizochytrium and mixtures thereof according to the invention in combination with an additive selected from the group consisting of rapeseed, flaxseed, soybean and avocadomile Is being generated. The advantage of this example is that the food product contains both short chain omega-3 HUFAs from the additive and long chain omega-3 HUFAs from the microflora. Foods with flaxseed, rapeseed, soybeans and avocado meal provide a source of short chain omega-3 HUFAs and can be extended by humans and animals ingesting short chain omega-3 HU.
It is well known to be useful in providing additional sources of FAs. However, there is a problem that such foods contain a large amount of choline from the added components, form primary amines, and may give an unpleasant odor to fish. Furthermore, when the concentration of toxic compounds from the additive components is high, it is possible that, for example, egg laying in chickens is suppressed, or animals do not accept food. Thus, the food of the present invention has the advantage of being able to reduce flaxseed, rapeseed, soybean or avocado meal content. This is because organisms ingesting food do not require high concentrations of short chain omega-3 HUFAs to convert to long chain HUFAs. Therefore, the amount of choline and / or inhibitory toxic compound in the food is reduced by reducing the content of flaxseed and rapeseed in the food.

The amount of the genus Thraustochytrium, the genus Schizochytrium and mixtures thereof used in foodstuffs is about 5 to 5.
It is in the range of 95% by weight. About 5-9 additional ingredients
It can be present in foods in the range of 5% by weight. In addition, the food product may also contain other ingredients, including grains, nutritional supplements, vitamins, binders and preservatives.

In a preferred embodiment, the food product described above is produced using extrusion. The microflora and the additive component are mixed by extrusion, and the humidity in the microflora biomass is reduced by the amount of the mixed additive component. The food is heated and extruded, further removing moisture from the food. The resulting product with a low water content can be dried naturally or by calcination for a relatively short time, the total energy required for drying and the degree of degradation of the Omega-3 HUFAs due to the high temperature for a long time. Lower. In addition, the heat generated from the extrusion can degrade some of the unwanted toxic compounds commonly found in the additive components. This toxic compound may, for example, prevent egg laying in chickens or the animals may refuse food.

The invention will be described in more detail according to examples. Species that meet the above selection criteria have not been described in the prior art. By using these selection criteria, more than 25 potentially promising strains were isolated from approximately 1,000 screened samples. About 20,50 in the American Type Culture Collection (ATCC)
It was later confirmed that among the 0 strains, 10 strains belonged to the same taxon when separated. Still viable strains were obtained at this repository and used to compare with strains isolated and cultured by the disclosed treatment. The results of this comparison are presented in Examples 4 and 5 below.

[0038] Recent classification theorists classify Thraustochydrids as algae or algae-like protists. All strains of the unicellular microorganisms disclosed and claimed herein are Thorstochriales (Th
raustochytriales) (Eyes: Thraustochytriales, Family: Thraustochytriales)
ytriaceae), genus: Thraustochytrium or Schizochytrium). For the sake of general description, these microorganisms will be referred to herein as microflora, as they more clearly indicate that the exact classification position is indeterminate.

The following new strains were deposited under the "Budapest Treaty on International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure": All restrictions on the public availability of the deposited material are removed at the same time as the patent is granted and cannot be revoked. Each deposit will be retained for at least 5 years after a recent application to provide a sample of deposited microorganisms has been accepted by the American Type Culture Collection (ATCC), and in any case at least 30 years after the date of deposit. .

The preferred microorganisms of the present invention are characterized by the fact that they are deposited strains, in particular they are capable of producing the Omega-3 HUFAs described herein, and under the conditions described herein. It has all the identifying characteristics of having the dimensional characteristics of cell aggregates when cultured. Specifically, the preferred microorganisms of the present invention refer to the following deposited microorganisms and mutants thereof.

Strain ATCC No. Date of deposit Schizochytrium S31 20888 8/8/88 Schizochytrium S8 20889 8/8/88 Although the present invention is disclosed with respect to a particular microbial strain, it was obtained on the basis of the disclosed suggestions. It includes all useful methods and strains available, including all alternatives, modifications and optimizations that are possible to those skilled in the art.

The following examples and test results are for purposes of illustration and are not intended to limit the scope of the invention. (Example) (Example 1. Collection and selection) 150m from the inland shallow salt water pond
l of sample water was collected and stored in a sterile polyethylene bottle.
Particular care was taken to also include some living plant material and naturally occurring organic deposits (septic and plant material) with the sample water. The sample was placed on ice until it was returned to the laboratory. The sample water was shaken in the laboratory for 15-30 seconds and 1-10 ml of this sample was pipetted, i.e. injected into a filter device with two filters. These filters are 1) a sterile 47 mm Whatman No. 4 filter with a pore size of about 25 μm at the top and 2) a 47 mm diameter polycarbonate filter with a pore size of about 1.0 μm at the bottom of the Whatman filter. Small variations in the nominal size of the filter result in cell sizes collected on the polycarbonate filter in the range of about 1.0-25 μm.

Remove Whatman filter and discard
It was Polycarbonate filter with solid F inside Petri plate
-1 was placed on the medium. This medium is 6 (per liter)
00 ml seawater (artificial seawater available), 400 ml
Distilled water, 10g agar, 1g glucose, 1g tampa
Hydrolyzate, 0.2 g yeast extract, 2 ml of 0.
1M KH₂PO_Four1 ml vitamin solution (A-vit
s) (100 mg / l thiamine, 0.5 mg / l biamine
Containing Ochin and 0.5 mg / l Cyanocobalamin
Yes), 5 ml of trace metal mixture (PII metal, 1 liter)
The content per unit is 6.0 g of Na₂EDTA, 0.29
g FeCl₃・ 6H₂O, 6.84g H₃BO ₃, 0.
86 g MnCl₂・ 4H₂O, 0.06g ZnC
l₂, 0.026 g of CoCl₂・ 6H₂O, 0.052
g of NiSO_Four・ H₂O, 0.002g CuSO_Four・ 5
H₂O and 0.005 g Na₂MoO_Four・ 2H₂O. )common
And 500 mg each of streptomycin sulfate and peni
It consists of Shirin-G. Agar plate in the dark at 30 ° C
The culture was incubated. After 2-4 days, many colonies on the filter
-Has appeared. Play single cell microflora (excluding yeast)
Picked up on a new plate with the same medium composition.
The streak culture was performed again. Colonies of almost colorless cells
Special care was taken to pluck everything. New plate
Incubate at 30 ℃ for 2 to 4 days,
One colony was picked. Next, pick a single colony
50 ml liquid culture containing enriched organics similar to agar plates
I put it on the ground. These medium strains are used in the rotary shaker
2 to 4 days at 30 ° C on a roll (100-200 rpm)
Incubation was carried out for a while. The medium strain appears to have reached maximum density
Then, 20-40 ml of this was collected and centrifuged.
And freeze dried. Next, clarify the fatty acid content of the strain
Is a well-known technique for gas chromatography
(Lepage and Roy, 1984
Years) analyzed the sample. Therefore, Omega 3 series
To identify strains containing HUFAs and to select them further
These medium strains were maintained.

Utilizing the collection and selection methods outlined above, more than 150 single cell microflora strains were isolated. These strains have a high omega-3 HUFA content as a percentage of total fatty acids and grow at temperatures ranging from 15 to 48 ° C. Depending on the application, harmful C20:
Strains having less than 1% (as a percentage of total fatty acids) of 4n-6 and C22: 5n-6HUFAs can also be isolated. Strains with high Omega-6 content can also be isolated. Strains of these microflora can be repeatedly isolated from the same location using the procedure outlined above. It is not uncommon for freshly isolated strains to have fatty acids that look very similar. At this point, it cannot be denied that there may be duplicate isolates of the same strain. Further screening can then be performed by similar methods to obtain other desirable properties such as salt tolerance or the ability to use a variety of carbon and nitrogen sources.

(Example 2. Maintenance of unlimited growth: PO ₄ and yeast extract) Schizochyt aggregatum (Schizochyt)
rium Aggregatum) (ATCC 28209) cells were harvested from solid F-1 medium and inoculated into 50 ml FFM medium (Fuller et al., 1964). This medium is 1,000 ml of seawater, 1.0 g of glucose, 1.0 g of gelatin hydrolyzate, 0.01 g of liver extract, 0.1 g of yeast extract, 5 ml of PII metal, 1 ml of B-.
Vitamin solution (Goldstein et al., 1
969) and 1 ml of antibiotic solution (25 g / l streptomycin sulfate and penicillin-G). 1.0 ml of vitamin compound (pH 7.2) contains 200 μg of thiamine HCl, 0.5 μg of biotin, 0.05 μg of cyanocobalamin, and 100 μ of nicotinic acid.
g, 100 μg of calcium pantothenate, 5.0 μg of riboflavin, 40.0 μg of pyridoxine HCl,
20.0 μg of pyridoxamine 2HCl, 10 μg of p-aminobenzoic acid, 500 μg of chlorine HCl, 1.0 mg of inositol, 0.8 g of thymine, 0.26 mg of orotic acid, 0.2 μg of folinic acid and 2.
Contains 5 μg. The culture was placed on a rotary shaker (200 rpm) at 27 ° C. After 3-4 days, 1 ml of this culture was transferred to 50 ml each of the following treatments. 1) FFM medium (as a control) and 2) 2
FFM medium supplemented with 50 mg / l KH ₂ PO ₄ and 250 mg / l yeast extract. 48 of these cultures
Rotation shaker (200 rpm)
Put it on top. Cells were harvested and cell yield was quantified.
In the first treatment, the final concentration of cells on an ashless dry weight basis was 616 mg / l. In the second treatment, the final concentration of cells was 1,675 mg / l, indicating that the effect of increasing the concentration of PO ₄ and yeast extract in the medium was emphasized.

Example 3. Maintaining unlimited growth: Corn steep liquor as a substitute for yeast extract Schizochytrium sp. Cells of S31 (ATCC No. 20888) were harvested from solid F-1 medium and placed in 50 ml of M-5 medium. 1g of this medium (on a 1-liter basis)
Yeast extract, 25g NaCl, 5g MgSO ₄ ·
7H ₂ O, 1 g KCl, 200 mg CaCl ₂ , 5 g
Glucose, 5 g glutamate, 1 g KH ₂ P
It consists of O ₄ , 5 ml PII metal, 1 ml A-vitamin solution and 1 ml antibiotic solution. The pH of this solution was adjusted to 7.0 and the solution was filter sterilized. A sterile solution of corn steep liquor (4 g / 40 ml; pH 7.0) and yeast extract (1 g / 40 ml; pH 7.0) was prepared. The following amounts of yeast extract solution were added to a set of M-5 medium flasks. 1) 2 ml; 2) 1.5 ml;
3) 1 ml; 4) 0.5 ml and 5) 0.25 ml. The yeast extract and corn steep liquor solution were added to another set of M-5 medium flasks at the following concentrations.
1) 2 ml yeast extract; 2) 1.5 ml yeast extract and 0.5 ml corn steep liquor; 3) 1.0
ml yeast extract and 1.0 ml corn steep liquor; 4) 0.5 ml yeast extract and 1.5 ml corn steep liquor and 5) 2 ml corn steep liquor. Only 1 ml of culture in F-1 medium was used to inoculate each flask. These cultures were placed on a rotary shaker for 48 hours at 27 ° C.
Cells were harvested by centrifugation and cell yield was measured (as ashless dry weight). The results are shown in Table 1.
This result indicates that the cell yield can be increased by adding the yeast extract to the medium of 0.8 g / l. However, the addition of corn steep liquor together with yeast extract is even more effective and doubles the yield of treating agent. Since corn steep liquor is much cheaper than yeast extract, this is a great advantage for economically producing cells.

[0047]

[Table 1] Example 4. Increasing the HUFA content of the strain isolated by the method of Example 1 when compared to the ATCC strain (known strain) A group of freshly isolated strains selected based on the method described in Example 1 151 strains were sampled during the late exponential growth phase and analyzed for HUFA content by gas chromatography. All strains are M1 medium or liquid FFM
Growing in either of the media showed the highest yield of cells in either case. The exception is that the M1 medium has the same composition as the M5 medium and the glucose and glutamate concentrations are 1 g / l. In addition, five previously isolated Thraustochytrium or Schizochytrium species were obtained from the American Type Culture Collection. This preservation institution lists all strains that can be obtained from preserves in a viable form. These strains are T. Aureum (ATCC No. 28211)
No.), T. Aureum (ATCC No. 34304),
T. Roseum (ATCC No. 28210),
T. Straitum (ATCC No. 34473)
No.) and S. Aggregate (ATCC No. 28209
It was No.). It was shown that growth of all strains was shortened in conventional media, and that growth was generally improved in media such as M5 medium and FFM medium of the present invention. Based on the improved growth of the strain in the medium of the present invention, the fatty acid production of each known strain was measured as described above.

Fatty acid peaks were identified by using pure compounds of known structure. Weighted by weight percent of total fatty acids by integrating the chromatographic peaks. The identified compounds are palmitic acid (C16: 0), C20: 4n-6 and C22: 1.
(These were not decomposed separately depending on the system used), C20: 5n-3, C22: 5n-6, C2.
It was 2: 5n-3 and C22: 6n-3. The remaining fatty acids, which are usually of low molecular weight, are grouped under the category of "other fatty acids". 20: 5n-3,2 of all omega-3 fatty acids
Calculated as the sum of 2: 5n-3 and 22: 6n-3. Total omega-6 fatty acids were calculated as the sum of the 20: 4/22: 1 peak and the 22: 5n-6 peak.

The results are shown in Tables 2-7 and FIGS. It can be seen from Tables 2-4 that a large number of strains can be isolated by the method of the present invention and that there are many strains superior to known strains by some important criteria. For example, 102 strains produce at least 7.8% by weight of total fatty acids of C20: 5w3, which has a higher proportion of fatty acids than any of the known strains. Strain 23B
(ATCC No. 20892) and 12B (ATCC No. 2)
No. 0890) is an example of this strain. The 30 strains of the present invention include at least 6 of the total fatty acids as omega-3 fatty acids.
It produced 8% by weight, which is higher than any known strain. Strain 23B (ATCC No. 20892) is an example of this strain. The 76 strains of the present invention produced only 10% by weight of total fatty acids as omega-6 fatty acids, which are considered undesirable for human nutrition, which is less than any known strain. Strain 23B (ATCC
No. 20892) and 12B (ATCC No. 20890)
No.) is an example of this strain. In addition, there are 35 strains of the invention that produce more than 25% by weight of total fatty acids as omega-6 fatty acids, more than any known strain. Although these strains can be said to have a relatively narrow utilization range for nutritional purposes, they are useful as a feedstock for the chemical synthesis of eicosanoids starting from omega-6 fatty acids.

In addition, the data reveal that there are many strains that produce a high percentage of total omega-3 fatty acids as C22: 6n-3. In Tables 5-7, Table 2-
48 of the strains shown in 4 were compared with known strains to obtain C2
0: 5n-3, C22: 5n-3 and C22: 6n-3
Each is shown as a total weight percentage of Omega-3 series. Fifteen strains had at least 94% by weight of total omega-3 fatty acids as C22: 6n-3, more than any known strain. Strain S8 (ATCC No. 208
89) was an example of this strain. 18 strains are C2
At least 2 of all omega-3 fatty acids as 0: 5n-3
8% by weight, which is more than any known strain. Strain 12B (ATCC No. 20890) was an example of this strain.

[0051]

[Table 2]

[0052]

[Table 3]

[0053]

[Table 4]

[0054]

[Table 5]

[0055]

[Table 6]

[0056]

[Table 7] FIG. 1 shows a series of strains isolated by the method in Example 1 having greater than 67% (percent total fatty acids) omega-3 fatty acids and less than 10.6% (percent total fatty acids) omega-6 fatty acids. Shows. All known strains had an omega-3 fatty acid below 67% (percent total fatty acids) and an omega-6 fatty acid above 10.6% (percent total fatty acids).

FIG. 2 shows that more than 67% (in percent total fatty acids) of omega-3 fatty acids and 7.5 (in percent total fatty acids) were separated by the method in Example 1.
1 shows a series of strains with greater than 20% C20: 5n-3. All known strains had an omega-3 fatty acid below 67% (percent total fatty acids) and a C20: 5n-3 below 7.8% (percent total fatty acids).

Example 5 Increase in growth rate of strain isolated by the method of Example 1 when compared with ATCC strain (well-known strain) Schizochytrium sp. S31 (ATCC No. 20
No. 888), Schizochytrium sp. S8 (ATCC No. 20889), Thraustochytrium sp. S4
2, Thraustochytrium sp. U42-2, Thraustochytrium sp. U42 and U30 and Thraustochytrium aureum (ATCC No. 28211)
And Schizochytrium aggregatum (ATCC No. 282
Cells of No. 09) (well-known strain) were picked from the solid F-1 medium and placed in 50 ml of M-5 medium. PH of this solution
Was adjusted to 7.0 and the solution was filter sterilized. After growing for 3 days on a rotary shaker (200 rpm, 27 ° C),
1-2 ml of each culture strain was transferred to another flask of M-5 medium and placed on the same shaker for 2 days. next,
These cultures (1-2 ml) were transferred to another flask of M-5 medium and placed on a shaker for 1 day. By this step all cultures were in exponential growth phase. Then, using these late cultures, two 2 of M-5 media were used.
It was decided to inoculate a 50 ml flask and grow a strain in each. The flasks were then placed on a shaking incubator at 25 ° C and 30 ° C and monitored for changes in their optical density on a Beckman DB-G spectrophotometer (660 nm, 1 cm path). Optical density reading is 0, 6, 10, 14,
It was carried out at 17.25, 20.25 and 22.75 hours. Next, the exponential growth rate (doubling per day) was calculated from the optical density data by the method of (Sorokin) 1973. The results are shown in Table 8 and FIG. 4 (growth of strain U30 at 25 ° C. is standard). The strain isolated by the method in Example 1 had a much higher growth rate than the known ATCC strain at both 25 ° C. and 30 ° C. and was optimized for the phosphate concentration essential for continuous growth. However, the data show that the results are the same. 30 strains of Thraustochytriales isolated from cold water in Antarctica
It showed no signs of growing at ° C.

[0059]

[Table 8] (Example 6. Improvement of production characteristics (growth and lipid induction) of the strain isolated by the method of Example 1 when compared with ATCC strain (conventional strain)) Schizochytrium sp. S31 (AT
CC No. 20888), Schizochytrium sp. S8
(ATCC No. 20889) (both separated by the method of Example 1) and Thraustochytrium aureum (ATCC
No. 2821 1) and Schizochytrium aggregatum (ATCC No. 28209) (conventional strain) cells were picked from solid F-1 medium and placed in 50 ml of M-5 medium (see Example 3). ). The pH of this solution was adjusted to 7.0 and the solution was filter sterilized. Rotation shaker (200r
pm, 27 ° C.) for 3 days,
2 ml was transferred to another flask of M-5 medium and placed on the same shaking incubator for 2 days. Next, the ashless dry weight of each of these cultures was quickly measured to obtain 50 ml of M-5.
3.29 mg of each culture was pipetted into two 250 ml Erlenmeyer flasks containing medium. The flasks were placed on a rotary shaker (200 rp).
m, 27 ° C). After 24 hours, 20 ml of each culture was centrifuged, the supernatant was discarded and 250 m with 50 ml of M-5 medium containing no glutamate (N source).
The cells were transferred to an Erlenmeyer flask. The flask was placed back on the shaker, Ropage and Roy (1984).
After 12 hours, the ashless dry weight was measured by the above method to quantify the fatty acid content. The results are shown in FIG. 5 (the yield of ATCC No. 28211, which is a known strain, is standard)
Is shown in. The results show that the strain isolated by the method of Example 1 produced 2-3 times more ashless dry weight than the conventional ATCC strain within the same time under exponential growth and nitrogen limitation. In addition, the yields of total fatty acids and omega-3 fatty acids obtained from the strain of the present invention are increased, and strain S31 (ATCC No. 20888) is a conventional A
It produces 3-4 times more omega-3 fatty acids than the TCC strain.

Example 7 Improved Salt Tolerance and Fatty Acid Production by Strains Isolated by the Method of Example 1 Four strains of Thraustochytrid, Schizochytrium sp. S31
(ATCC No. 20888), Thraustochytrium sp. U42-2 (ATCC No. 20891) (both separated and sorted by the method of Example 1) and S. Aggregatem (ATCC No. 28209) and T.W. Aureum (A
TCC No. 28210) (obtained from American Type Culture Collection) was harvested from the solid F-1 medium and incubated at 27 ° C. for 3 to 4 days on a rotary shaker (200 rpm). M medium salt (2
5 g / l NaCl, 5 g / l MgSO ₄ .7H ₂ O,
Media with different salinity ranges were prepared by diluting 1 g / l KCl, 200 mg / l CaCl ₂ ).
It is 1) 100% (w / v) M medium salt, 2) 80
% (V / v) M medium, 20% (v / v) distilled water,
3) 60% (v / v) M medium, 40% (v / v) distilled water, 4) 40% (v / v) M medium, 60% (v /
v) distilled water, 5) 20% (v / v) M medium, 80%
(V / v) distilled water, 6) 15% (v / v) M medium,
85% (v / v) distilled water, 7) 10% (v / v) M
Medium, 90% (v / v) distilled water, 8) 7% (v / v)
M medium, 93% (v / v) distilled water, 9) 3% (v / v)
v) M medium, 97% (v / v) distilled water, 10) 1.
5% (v / v) M medium, 98.5% (v / v) distilled water. The following nutrients: 5 g glucose, 5 g glutamate, 1 g yeast extract, 200 mg (N
H ₄ ) ₂ SO ₄ , 200 mg NaHCO ₃ , 5 ml PI
Metal I, 1 ml A vitamin solution and 2 ml antibiotic solution were added to the treatment (per liter). 50 ml of each of these treatment agents was inoculated with 1 ml of cells growing in F-1 medium. These cultures were placed on a rotary shaker (200 rpm) and maintained at 27 ° C for 48 hours. Cells were harvested by centrifugation and total fatty acids were measured by gas chromatography. The result is shown in FIG. Thraustochytrium sp. Isolated by the method of Example 1 U42-2 (ATCC No. 20891) is a T.U.
Almost twice the amount of fatty acids produced by Aureum (ATCC No. 28211), S. Aggregate (ATCC No. 2
No. 8209) can produce more than 8 times the amount of fatty acid produced. In addition, U42-2 appears to have a broader range of salt resistance at the upper end of the evaluated salinity range.
Also isolated by the method of Example 1, Schizochytrium sp.
S31 (ATCC No. 20888) had a high fatty acid yield (2.5 to 10 times that of the known ATCC strain), and exhibited a far broader range of salt tolerance than the ATCC strain. In addition, Schizochytrium sp. S31 (ATCC No. 20888) is maximally grown at very low salinity. Considering the marketability, this property is economically significant. that is,
This is because the salt water has a corrosive effect on the metal reactor and causes problems associated with salt water treatment.

Example 8 Culture / Low Salinity 50 ml of M / 10-5 medium in a 250 ml Erlenmeyer flask was added to Schizochytrium sp. A colony of S31 (ATCC No. 20888) was inoculated. M / 10-5 medium is 1000 ml deionized water, 2.5 g NaCl,
MgSO ₄ · 7H ₂ O of 0.5 g, 0.1 g of KCl,
0.02 g CaCl ₂ , 1.0 g KH ₂ PO ₄ , 1.
0 g yeast extract, 5.0 g glucose, 5.0 g glutamic acid, 0.2 g NaHCO ₃ , 5 ml PII
It contains trace metals, 2 ml of vitamin formulation and 2 ml of antibiotic formulation. Rotation shaker (200 rp
The culture was incubated at 30 ° C. on m). After 2 days, the culture had a moderate density and was actively growing. 20 ml of this actively growing culture is used to
A 2 liter fermenter containing 700 ml was inoculated. However, in this medium, the concentrations of glucose and glutamate were increased to 40 g / l (M / 10-40 medium). Aeration of fermented soybean at 30 ℃, 1vol / min,
Maintained at 300 rpm mixing. After 48 hours, the cell density in the fermenter was 21.7 g / l. Cells were harvested by centrifugation, lyophilized and stored under N ₂ .

The total fatty acid content and the omega-3 fatty acid content were measured by gas chromatography. The total fatty acid content of the final product was 39.0% ashless dry weight. Omega-3 FUFA content of microbial products (C20: 5n
-3, C22: 5n-3 and C22: 6n-3) were 25.6% of the total fatty acid content. The ash content of the sample is 7.0%
Met.

Example 9 Diversity of Fatty Acid Content Growth and gas chromatographic analysis of fatty acids produced by the various strains described in Example 4 revealed differences in fatty acid diversity. The strain of the present invention synthesizes different fatty acids less than the strains obtained conventionally. Low fatty acid diversity is more effective in the purification of fatty acids, as less impurities have to be separated. Since the possibility of ingesting unnecessary fatty acids is reduced for the purpose of supplementing food, it is more effective that the number of fatty acids different from each other is small. Table 9 shows the number of HUFAs which are present in the well-known strains designated by the ATCC number and various strains of the present invention in a concentration of 1% or more in total fatty acid weight, and which are different from each other.

[0064]

[Table 9] (Example 10. Recovery) 50 ml of M5 medium in a 250 ml Erlenmeyer flask was added to Schizochytrium sp. A colony of S31 (ATCC No. 20888) was inoculated. 30 on a rotary shaker (200 rpm)
The culture was incubated at 0 ° C. After 2 days, the culture had a moderate density and was actively growing. Using 20 ml of this actively growing culture, 1000 ml of the same medium
The contained 1 liter fermented soybean was inoculated. However, in this medium, the concentration of glucose and glutamate was 40 g / l.
Was increased (M20 medium). Fermented soy sauce at 30 ℃, p
Maintained at H7.4 and aeration at 1 vol / mi
n, mixing was 400 rpm. After 48 hours, the cell density in the fermentor was 18.5 g / l. Within 2-4 minutes after stopping aeration and mixing in the fermentor, the cells aggregated and precipitated in 250 ml of the lower part of the fermentor. This aggregation area of cells had a cell density of 72 g / l. This cell range can be siphoned from the fermentor and (1) transferred to another reactor during the nitrogen-limited time (eg, combining high density production of several fermenters) or (2) centrifugation. Alternatively, it can be directly collected by filtration. By preaggregating the cells in this manner, the amount of water that needed to be processed to recover the cells was reduced by 60-80%.

Example 11. Utilization of various carbon and nitrogen sources 50 ml of M5 medium in 250 ml Erlenmeyer flask
, Schizochytrium sp. Collected from the agar slope. S3
1 (ATCC No. 20888) or Thraustochytrium sp. U42-2 (ATCC No. 20891) colonies were inoculated. The M5 medium is described in Example 3,
The difference lies in the addition of 2 ml of the vitamin compound and 2 ml of the antibiotic mixture. The culture strain was incubated at 30 ° C. on a rotary shaker (200 rpm). After 2 days, the culture had a moderate density and was actively growing. Using this culture, a flask of M5 medium is inoculated with one of dextrin, sorbitol, fructose, lactose, maltose, sucrose, corn starch, wheat starch, potato starch, and ground corn as an alternative to glucose (( 5 g /
l), or one of gelysate, peptone, tryptone, casein, corn steep liquor, urea, nitrate, ammonium, whey or corn gluten meal as an alternative to glutamate (5 g / l). Rotation shaker (200rpm, 27
The culture was incubated at 48 ° C for 48 hours. Tables 10 and 11 show the culture densities comparing the growth on different organic substances.
Is shown in.

[0066]

[Table 10]

[0067]

[Table 11] Example 12. Feeding of a Thraustochytrid-based feeding supplement for the purpose of increasing the omega-3 HUFA content of saltwater shrimp. Thraustochytrium sp. Sp. In a shake flask of M-5 medium. Cellular biomass of 12B (ATCC No. 20890) was produced at 25 ° C (see Example 3). In a shake flask of M / 10-5 medium, Thraustochytrium sp. S31 (ATC
Cellular No. C 20888) was produced at 27 ° C (see Example 8). The cells of each strain were collected by centrifugation. The pellet was washed once with distilled water and centrifuged again to produce a 50% solid paste. The resulting paste was resuspended in seawater and then added to the culture for adult saline shrimp as a feed supplement. The saltwater shrimp were preliminarily bred on old agricultural products, and as a result, the saltwater shrimp had a very low omega-3 HUFA content, only 1.3-2.3% of the total fatty acids (wild saltwater shrimp omega). The average content of the ternary HUFA is 6 to 8% of all fatty acids.). Keep saltwater shrimp (2-3 / ml) in a 1 liter beaker filled with seawater and use air stone (airsto
ne) was used to aerate and mix the culture. After addition of the feed supplement, saline shrimp samples were occasionally collected and washed and the fatty acid content was determined by gas chromatography. The results are shown in FIGS. 7 and 8. Feeding with a Thraustochytrid-based feeding supplement as the last feeding, the omega-3 content of the saltwater shrimp is within 5 hours when feeding strain 12B or within 11 hours when feeding S31 In addition, the omega-3 content of wild saltwater shrimp can be increased. Omega 3 HU of saltwater shrimp
FA content can be significantly increased over the omega-3 content of wild saline shrimp when fed with these supplements for up to 24 hours. In addition, these feeding supplements significantly increase the DHA content of saltwater shrimp. Usually, DHA has been reported only in trace amounts in wild saline shrimp.

Example 13 Use of Sodium Sulfate in the Medium In this example, when sodium sulfate was used as the sodium salt in the fermentation medium instead of sodium chloride, omega-3 system production and total fatty acid content were not impaired, and were equal. Or it has shown that it is excellent. Schizochytrium ATCC No. 20888 in a medium containing 2.36 g of sodium, 1.5 to 3.0 g of a nitrogen source and 3.0 g of glucose per liter of a medium, and having a pH of 7.0.
The number grew. The cells were incubated at 28 rpm at 200 rpm for 48 hours. The results are shown in Table 12.

[0069]

[Table 12] As can be seen from Table 12, the formation of omega-3 and total fatty acids when using sodium sulfate as the sodium salt is comparable or better when using sodium chloride.

Example 14 Production of Schizochytrium in a Low Salinity Medium In this example, fermentation of a Schizochytrium genus in a low salinity medium while maintaining a high yield of biomass and production of Omega-3 and fatty acids was carried out. Shows.

3.33 g / l peptone as a nitrogen source,
Schizochytrium ATCC No. 20888 was grown in a medium containing 5.0 g / l of glucose as a carbon source and having various sodium concentrations. About 40mg / l
The cells were fermented at 30 ° C. for 48 hours with the dwt inoculum. Sodium was supplied as sodium chloride. The progress results are shown in Table 13.

[0072]

[Table 13] As can be seen from the results in Table 13, the biomass yield and the production of omega-3 fatty acids and total fatty acids can be increased at a sodium concentration of about 1.0 g / l or more. Example 15. Cultivation of Schizochytrium in a medium with a low chloride content In this example, of the microflora of the present invention, which had a minimum chloride concentration but increased biomass yield based on the initial sugar concentration. Shows fermentation.

2 in 50 ml aliquots of the media shown below
Schizochytrium sp. ATCC No. 20888 was cultured in shake flasks at 00 rpm and 28 ° C. This medium contained 1000 ml deionized water, 1.2 g MgSO ₄ .7H ₂ O, 0.067 g CaCO ₃ , 3.
0 g glucose, 3.0 g monosodium glutamate, 0.2 g KH ₂ PO ₄ , 0.4 g yeast extract,
It consisted of 5.0 ml PII metal, 1.0 ml vitamin formulation and 0.1 g each of penicillin G and streptomycin sulfate. The chloride concentration was varied by varying the amount of KCl added to each treatment. The potassium concentration in all treatments was kept constant by adding potassium citrate. The sodium concentration was either 2.37 g / l or 4.0 g / l with the addition of sodium sulfate. The results of these fermentations are shown in Table 14 below.

[0074]

[Table 14] As can be seen from the results shown in Table 14, the biomass yield per sugar can be increased at a low chloride concentration. For example, a yield of 50% or more of the theoretical value is achieved at a chloride concentration of 59.1 mg / l or more.

Example 16 Variation of Sodium Sulfate Concentration at Low Chloride Concentration This example shows the effect of various sodium sulfate concentrations on fermentation at low chloride concentration.

2 in 50 ml aliquots of the media shown below
Schizochytrium sp. ATCC No. 20888 was cultured in shake flasks at 00 rpm and 28 ° C. This medium contains 1000 ml deionized water, 1.2 g MgSO ₄ .7H ₂ O, 0.125 g KCl, 0.06.
7 g CaCO ₃ , 3.0 g glucose, 3.0 g monosodium glutamate, 0.2 g KH ₂ PO ₄ ,
0.4 g yeast extract, 5.0 ml PII metal, 1.
It consisted of 0 ml vitamin formulation and 0.1 g each of penicillin G and streptomycin sulfate. The concentration of sodium sulfate in the treating agent is 3.0 to 30.2 g /
changed to 1. The results of fermentation progress are shown in Table 15 below.

[0077]

[Table 15] The results shown in Table 15 show that, at a low chloride concentration of about 59 g / l, by selecting an appropriate sodium sulfate concentration, glucose can yield a large biomass yield of 50% or more of the theoretical value.

[0078]

As described in detail above, according to the present invention,
The microflora and the method of growing the same can be modified to produce high concentrations of highly unsaturated omega-3 fatty acids.

[Brief description of drawings]

FIG. 1 is a graphic representation of HUFA production in a newly isolated strain of the invention (●) and a previously isolated strain (+). Each point represents a strain, and the position of each point is determined by the weight percentage of the total fatty acids of the omega-3 system HUFAs (abscissa) and the weight percentage of the omega-6 system fatty acids (ordinate) of the total fatty acid. In the strain of the present invention, 10.6% (w / w) or less of total fatty acids were shown to be in the omega-6 system, and 67% or more of total fatty acids were shown to be in the omega-3 system.

FIG. 2 is a graphic representation of HUFA production in a newly isolated strain of the invention (●) and a previously isolated strain (+). Each point represents a strain, and the position of each point is the weight percentage of all fatty acids of omega-3 HUFAs (abscissa) and eicosapentaenoic acid (EPA C20: 5n-).
3) Determined by the weight percentage of total fatty acids (ordinate). The strain of the present invention comprises 67% of all fatty acids
(W / w) and above are Omega 3 series, 7.8% of total fatty acids
The above (w / w) was shown only within the range of C20: 5n-3.

FIG. 3 is a graph of Omega-3 HUFA composition in the newly isolated strain of the present invention (□) and the previously isolated strain (+). Each dot represents an individual strain. The abscissa value is the weight fraction of C20: 5n-3 total omega-3 HUFAs and the ordinate value is the weight fraction of C22: 6n-3 total omega-3 highly unsaturated fatty acids. Only the strain of the present invention has a C20: 5n-3 weight fraction of 28% or more, or a C22: 6n-3 weight fraction of 93.6%.
Was shown in the range above.

FIG. 4 is a graph showing growth at 25 ° C. and 30 ° C. of various freshly isolated strains of the present invention and previously isolated strains. The growth rate is normalized to that of strain U-30 at 25 ° C. Previously isolated strains are designated by ATCC accession number.

FIG. 5 is a graph of total yield of cell production after induction with nitrogen limitation. Each of the ashless dry weights, total fatty acids, and omega-3 HUFAs as shown was shown to have the corresponding value of strain 28211 as the standard. All strains are ATC
C It is identified by the accession number.

FIG. 6 is a graph of fatty acid yield after growth in media with salinity shown on the abscissa. The illustrated strain is the newly isolated strain S31 (ATCC 20888).
(□) and U42-2 (ATCC 20891) (+)
And the previously isolated strain ATCC 28211
(⋄) and ATCC 28209 (△). The fatty acid yield was 1.0 based on the average growth rate of S31 (ATCC 20888) (□) over the test salinity range.
Plotted as relative yield with 0 as standard.

FIG. 7 shows the increase in the content of Omega-3 HUFA in the total lipids of saltwater shrimp called Artemia salina fed with the Thraustochytrid strain (ATCC 20890) isolated by the method of Example 1. It is a graph shown. EPA is C20: 5n-3,
DHA is C22: 5n-3.

FIG. 8: Omega-3 strain H in total lipids in saltwater shrimp Artemia salina fed a Thraustochytrid strain (ATCC 20888) isolated by the method of Example 1.
It is a graph which shows the increase of UFA content. EPA is C2
0: 5n-3, DHA is C22: 5n-3.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1:90) C12R 1:89 (C12N 1/12 C12R 1:89) (58) Fields investigated (Int.Cl. ⁷ , DB name) A23L 1/28 C12N 1/10 C12N 1/12 BIOSIS / WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A ) long-chain omega-3 highly unsaturated fatty acid
Contains, and the genus Thraustochytriales
A microorganism selected from the genus Urum and a mixture thereof, and b) containing a short-chain omega-3 highly unsaturated fatty acid,
, Flaxseed, rapeseed, soybean, avocado meal and these
A food product comprising a substance selected from the group consisting of a mixture. 2. The food contains about 5 to 95% by weight of the substance.
The food according to claim 1, which comprises: 3. The method according to claim 1, wherein the food is an extruded product.
Listed food. 4. A ) long-chain omega-3 highly unsaturated fatty acid
Contains, and the genus Thraustochytriales
Obtained from microorganisms selected from the genus Urum and their mixtures
A fatty acid that is, b) containing omega-3 highly unsaturated fatty acid short chain, or
, Flaxseed, rapeseed, soybean, avocado meal and these
Fat obtained from a material selected from the group consisting of mixtures
A food consisting of acid.